Medicine containing genetically modified antibody against chemokine receptor ccr4

ABSTRACT

A medicament having a higher therapeutic effect than that provided by administration of a recombinant antibody against human CC chemokine receptor 4 or an antibody fragment thereof or an agent alone is provided.

TECHNICAL FIELD

The present invention relates to a medicament comprising a combinationof a recombinant antibody which specifically binds to human CC chemokinereceptor 4 (CCR4) or an antibody fragment thereof and at least oneagent.

BACKGROUND ART

When a ligand is bound to a chemokine receptor, migration of leukocytesis induced. Human CC chemokine receptor 4 (hereinafter referred to asCCR4) which is mainly expressed on a Th2-type CD4-positive helper T cellin a normal tissue is one type of a chemokine receptor family [Int.Immunol., 11, 81 (1999)]. CCR4 binds specifically to TARC (thymus andactivation-regulated chemokine) or MDC (macrophage-derived chemokine).The Th2-type CD4-positive helper T cell which controls humoral immunityis considered to play an important role in allergic diseases orautoimmune diseases.

In T cell-type leukemia/lymphoma cells described above, variouschemokine receptors are expressed, and there is a relation betweensubtypes of T cell leukemia/lymphoma and types of receptors expressed incells. It was reported that CCR4 is expressed at high frequency inleukemia/lymphoma cells [Blood, 96, 685 (2000)]. Since CCR4 is expressedat high frequency in ALK-positive anaplastic large-cell lymphoma andmycosis fungoides, a possibility of CCR4 being a tumor marker havingquite a high selectivity in specific carcinomas was suggested [Blood,96, 685 (2000), Mod. Pathol., 15, 838 (2002), J. Invest. Dermatol., 119,1405 (2002)]. It was reported that CCR4 is expressed at quite a highfrequency also in adult T-cell leukemia (hereinafter referred to as ATL)caused by infection with human T-cell leukemiavirus type I) [Blood, 99,1505 (2002)]. Regarding the expression of CCR4 in ATL, the expression ofCCR4 significantly correlates with bad prognosis [Clin. Cancer Res., 9,3625 (2003)]. Further, CCR4 is selectively expressed in cells of chronicT cell lymphoma (hereinafter referred to CTL) [J. Invest. Dermatol.,119, 1405 (2002)].

Method for treating leukemia/lymphoma is mainly chemotherapy using acombination of plural low-molecular anticancer agents. However,chemotherapy that provides satisfactory therapeutic effects has been sofar unknown [Gan to Kagaku Ryoho, 26, Supplement I, 165-172 (1999)].

Among the CCR4-positive leukemia/lymphoma described above, prognosis ofATL is poor in particular. Concerning patients who suffer from acute orlymphatic leukemia occupying more than 70% of total ATL and haveexperienced common CHOP therapy (therapy using cyclophosphamide,vincristine, doxorubicin and prednisone in combination), 4-year survivalrate is approximately 5% [British J. Haematol., 79, 428-437 (1991)].

In usual chemotherapy, it is sometimes difficult to induce remissionbecause of advent of drug-resistant tumor cells or the like. However,excellent therapeutic results are sometimes obtained by combination ofchemotherapy and an antibody. Anti-HER2/neu humanized antibody rhuMAbHER2 (Herceptin/trastuzumab, Roche) exhibited an outstanding effectagainst breast cancer in combination therapy with a taxane anticanceragent [Clinical Therapeutics, 21, 309 (1999)]. Anti-CD20 human chimericantibody IDEC-C2B8 (Rituxan/rituximab, IDEC) exhibited an outstandingeffect against B cell lymphoma by combination therapy with multiple drugtherapy [J. Clin. Oncol., 17, 268 (1999)].

Combination therapy using an antibody and a cytokine is also expected asnew immunotherapy against tumors. A cytokine is a general term forvarious humoral factors that control intracellular interaction in animmune reaction. An antibody-dependent cell-mediated cytotoxic activity(hereinafter referred to as ADCC), one of cytotoxic activities, isinduced by binding an antibody to an effector cell such as a mononuclearcell, a macrophage or an NK cell [J. Immunol., 138, 1992 (1987)]. Forthe purpose of activating an effector cell, combination therapy using acombination of an antibody and a cytokine has been attempted. Withrespect to B cell leukemia/lymphoma, a clinical test administratingIDEC-C2B8 and interleukin (IL)-2 [British J. Haematol., 117, 828-834(2002)] or IDEC-C2B8 and granulocyte-colony stimulating factor[Leukemia, 17, 1658-1664 (2003)] in combination has been conducted.However, an outstanding therapeutic effect has not been observed incomparison with use of the antibody alone.

Anti-CCR4 antibody KM2760 has been known as a therapeutic agent againstthe CCR4-positive leukemia/lymphoma that selectively reduces tumor cellsvia ADCC (WO 03/18635). Combined use of an anti-CCR4 antibody and achemotherapeutic agent or a cytokine has been unknown so far.

In treatment of cancers, especially, leukemia and lymphoma, atherapeutic method that brings forth satisfactory effects has beenunknown so far.

DISCLOSURE OF THE INVENTION

The object of the present invention is to provide a medicamentcomprising a combination of anti-CCR4 recombinant antibody or anantibody fragment thereof and at least one agent.

The present invention relates to the following (1) to (26).

(1) A medicament comprising a combination of a recombinant antibodywhich specifically binds to human CC chemokine receptor 4 (CCR4) or anantibody fragment thereof and at least one agent.

(2) A medicament for administering a combination of a recombinantantibody which specifically binds to CCR4 or an antibody fragmentthereof and at least one agent.

(3) A medicament for administering a recombinant antibody whichspecifically binds to CCR4 or an antibody fragment thereof and at leastone agent either simultaneously or successively.

(4) The medicament according to any one of the above (1) to (3), whichis an antitumor drug.

(5) The medicament according to the above (4), wherein the tumor is atumor in which CCR4 is expressed.

(6) The medicament according to the above (5), wherein the tumor inwhich CCR4 is expressed is a hematopoietic organ tumor.

(7) The medicament according to any one of the above (1) to (6), whereinthe recombinant antibody which specifically binds to CCR4 or theantibody fragment thereof is an antibody which specifically binds to anextracellular region of CCR4 and does not show a reactivity to a humanplatelet.

(8) The medicament according to the above (7), wherein the recombinantantibody which specifically binds to the extracellular region of CCR4 orthe antibody fragment thereof does not have an activity of inhibitingbinding of TARC (thymus and activation-regulated chemokine) or MDC(macrophage-derived chemokine) as a CCR4 ligand to CCR4.

(9) The medicament according to the above (7) or (8), wherein theextracellular region is an extracellular region selected from the groupconsisting of 1 to 39, 98 to 112, 176 to 206 and 271 to 284 of an aminoacid sequence represented by SEQ ID No. 1.

(10) The medicament according to any one of the above (7) to (9),wherein the extracellular region is an epitope existing at positions 2to 29 of the amino acid sequence represented by SEQ ID No. 1.

(11) The medicament according to any one of the above (7) to (10),wherein the extracellular region is an epitope existing at positions 13to 29 of the amino acid sequence represented by SEQ ID No. 1.

(12) The medicament according to any one of the above (7) to (11),wherein the extracellular region is an epitope existing at positions 13to 25 of the amino acid sequence represented by SEQ ID No. 1.

(13) The medicament according to the above (12), wherein in therecombinant antibody which specifically binds to CCR4 or the antibodyfragment thereof, a binding activity to a peptide comprising 13 to 25 ofthe amino acid sequence represented by SEQ ID No. 1 in which at leastone of tyrosine residues 16, 19, 20 and 22 is sulfated is lower than abinding activity to a peptide comprising 13 to 25 of the amino acidsequence represented by SEQ ID No. 1.

(14) The medicament according to any one of the above (1) to (13),wherein the recombinant antibody which specifically binds to theextracellular region of CCR4 or the antibody fragment thereof is anantibody which specifically reacts with an epitope recognized by amonoclonal antibody which hybridoma KM2160 (FERMBP-10090) produces or anantibody fragment thereof.

(15) The medicament according to any one of the above (1) to (14),wherein the human recombinant antibody is a human chimeric antibody or ahuman CDR-grafted antibody.

(16) The medicament according to the above (15), wherein the humanchimeric antibody comprises complementarity determining regions (CDRs)of a heavy chain (H chain) variable region (V region) and a light chain(L chain) V region of a monoclonal antibody which specifically binds toCCR4.

(17) The medicament according to the above (15) or (16), wherein thehuman chimeric antibody comprises CDR1, CDR2 and CDR3 of a heavy chain(H chain) variable region (V region) of an antibody comprising aminoacid sequences represented by SEQ ID Nos. 5, 6 and 7 and/or CDR1, CDR2and CDR3 of a light chain (L chain) variable region (V region) of anantibody comprising amino acid sequences represented by SEQ ID Nos. 8, 9and 10, respectively.

(18) The medicament according to the above (15) to (17), wherein thehuman chimeric antibody comprises a heavy chain (H chain) variable,region (V region) of an antibody molecule comprising an amino acidsequence represented by SEQ ID No. 11 and/or a light chain (L chain) Vregion of an antibody molecule represented by SEQ ID No. 12.

(19) The medicament according to the above (15), wherein the humanCDR-grafted antibody comprises complementarity determining regions(CDRS) of a heavy chain (H chain) variable region (V region) and a lightchain (L chain) V region of a monoclonal antibody which specificallybinds to CCR4.

(20) The medicament according to the above (15) or (19), wherein thehuman CDR-grafted antibody comprises CDR1, CDR2 and CDR3 of a heavychain (H chain) variable region (V region) of an antibody comprisingamino acid sequences represented by SEQ. ID Nos. 5, 6 and 7 and/or CDR1,CDR2 and CDR3 of a light chain (L chain) V region comprising amino acidsequences represented by SEQ ID Nos. 8, 9 and 10, respectively.

(21) The medicament according to any one of the above (15), (18) and(20), wherein the human CDR-grafted antibody comprises a heavy chain (Hchain) variable region (V region) of an antibody molecule comprising anamino acid sequence represented by SEQ ID No. 16 or 17 and/or a lightchain (L chain) V region of an antibody molecule represented by SEQ IDNo. 18.

(22) The medicament according to any one of the above (1) to (21),wherein the agent is a protein or an agent having low-molecular weight.

(23) The medicament according to the above (22), wherein the protein isa cytokine or an antibody.

(24) The medicament according to the above (23), wherein the cytokine isa cytokine selected from G-CSF, M-CSF, interferon-α, IL-2 and IL-15.

(25) The medicament according to any one of the above (1) to (24),wherein the agent having low-molecular weight is a chemotherapeuticagent or a hormone therapeutic agent.

(26) The medicament according to the above (25), wherein thechemotherapeutic agent is an agent selected from vincristine,cyclophosphamide, etoposide and Methotrexate.

Examples of the medicament in the invention include a medicamentcomprising a combination of the recombinant antibody which specificallyreacts with CCR4 or the antibody fragment thereof and at least oneagent, a medicament for administering the recombinant antibody whichspecifically reacts with CCR4 or the antibody fragment thereof and atleast one agent in combination, and a medicament for administering therecombinant antibody which specifically reacts with CCR4 or the antibodyfragment thereof and at least one agent either simultaneously orsuccessively.

The medicament comprising a combination refers to a medicament in whichthe recombinant antibody which specifically binds to CCR4 or theantibody fragment thereof and at least one agent are prepared separatelyand these are administered in combination either simultaneously orsuccessively or a mixed medicament obtained by mixing each ingredient.The mixed medicament obtained by mixing each ingredient includes afusion antibody obtained by binding at least one agent to therecombinant antibody which specifically binds to CCR4 or the antibodyfragment thereof, and the like.

The recombinant antibody which specifically binds to CCR4 and theantibody fragment thereof in the invention (both of which are sometimesreferred to as an antibody of the invention) include a recombinantantibody which specifically reacts with an extracellular region of humanCCR4 and an antibody fragment thereof. A recombinant antibody which doesnot show a reactivity to a human platelet or an antibody fragmentthereof, a recombinant antibody having high ADCC or an antibody fragmentthereof, and the like are preferable.

That an antibody does not show a reactivity to a human platelet as herereferred to means that an antibody does not substantially reactive witha human platelet. Specifically, it means that a reactivity is not shownby the measurement with a flow cytometer.

Further, the antibody of the present invention include an antibody whichspecifically reacts with the region comprising positions 1 to 39, 98 to112, 176 to 206 or 271 to 284 in the amino acid sequence represented bySEQ ID NO:1; an antibody which specifically reacts with positions 2 to29 (SEQ ID NO:2) in the amino acid sequence represented by SEQ ID NO: 1is preferred, an antibody which specifically reacts with and positions12 to 29 (SEQ ID NO:3) in the amino acid sequence represented by SEQ IDNO: 1 is more preferred and an antibody which specifically reacts withpositions 13 to 25 (SEQ ID NO:4) in the amino acid sequence representedby SEQ ID NO:1 is most preferred. The antibody also includes an antibodywhich specifically reacts with an epitope recognized by a monoclonalantibody binding to CCR4 produced by hybidoma KM 2160 (FERM BP-10090).The hybridoma KM 2160 has been deposited with International PatentOrganism Depositary, National Institute of Advanced Industrial Scienceand Technology, Central 6, 1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki,Japan, on Aug. 12, 2004 with accession No. FERM BP-10090.

The antibody in the present invention is preferably an antibody lowbinding activity to a peptide in which at least one tyrosine residues atpositions 16, 19, 20 and 22 is sulfated in the peptide comprisingpositions 13 to 25 of the amino acid sequence represented by SEQ ID NO.1than a binding activity to a peptide comprising 13 to 25 of the aminoacid sequence represented by SEQ ID No. 1.

The antibody in the invention also includes an antibody produced bylectin-resistant cells recognizing a sugar chain structure in which1-position of fucose is bound to 6-position of N-acetylglucosamine inthe reducing end through α-bond in a complex type N-glycoside-linkedsugar chain (WO 02/31140, WO 03/85118 and WO 03/85107).

A recombinant antibody of the present invention includes a humanizedantibody, human antibody, and the like.

Examples of the humanized antibody are a human chimeric antibody, ahuman CDR-grafted antibody, and the like.

The human chimeric antibody refers to an antibody comprising H chain Vregion (hereinafter also referred to as HV or VH) of an antibody of anon-human animal, and L chain V region (herein after also referred to asLV or VL) of an antibody, and CH of human antibody and L chain C region(hereinafter also referred to as CL) of a human antibody. As thenon-human animal, any animal can be used so long as hybridomas can beprepared from the animal. Suitable animals include mouse, rat, hamster,rabbit and the like.

The human chimeric antibody of the present invention can be produced byobtaining cDNAs encoding VH and VL from a hybridoma capable of producinga monoclonal antibody derived from non-human animal which specificallyreacts with CCR4, inserting them into an expression vector for animalcell having genes encoding human antibody CH and human antibody CL tothereby construct a vector for expression of human chimeric antibody,and then introducing the vector into a host cell to express theantibody.

Any CH of a human chimeric antibody can be used, so long as it belongsto human immunoglobulin (hereinafter referred to as hIg), but those ofIgG class are preferred, and any one of subclasses further belonging toIgG such as γ1, γ2, γ3 and γ4 can be used. Also, as CL of a humanchimeric antibody, those of κ class or λ class can be used.

The human chimeric antibody of the present invention is a human chimericantibody which comprises CDR1, CDR2 and CDR3 of VH comprising the aminoacid sequences represented by SEQ ID NOs:5, 6 and 7, respectively, andCDR1, CDR2 and CDR3 of VL comprising the amino acid sequencesrepresented by SEQ ID NOs:8, 9 and 10, respectively. Specifically, itincludes a human chimeric antibody which comprises VH and VL comprisingthe amino acid sequences represented by SEQ ID NOs:11 and 12,respectively. More specifically, it includes a human chimeric antibodywherein the VH of the antibody consists of the amino acid sequencerepresented by SEQ ID NO: 11, the H chain C region of the human antibodyconsists of an amino acid sequence of the hIgG1 subclass, the L chain Vregion consists of the amino acid sequence represented by SEQ ID NO:12,and the L chain C region of the human antibody consists of an amino acidsequence of the κ class. An example includes anti-CCR4 human chimericantibody KM 2760 disclosed in WO01/64754.

The human CDR-grafted antibody refers to an antibody in which CDRs of VHand VL of an antibody derived from a non-human animal which specificallybinds to CCR4 are grafted into appropriate sites in VH and VL of a humanantibody.

The human CDR-grafted antibody of the present invention can be producedby constructing cDNAs encoding V regions in which CDRs of VH and VL of anon-human animal-derived antibody which specifically binds to CCR4 aregrafted into FR of VH and VL of an arbitrary human antibody, insertingthe resulting cDNAs into an expression vector for animal cells which hasDNAs encoding the CH and the H chain C region (hereinafter referred toas CL) of a human antibody, respectively, to construct a humanCDR-grafted antibody expression vector, and introducing the expressionvector into an animal cell to induce expression.

As the method for selecting the amino acid sequences of frameworks(hereinafter referred to as FR) of VH and VL of a human antibody, any ofthose derived from human antibodies can be used. Suitable sequencesinclude the amino acid sequences of FRs of VH and VL of human antibodiesregistered in databases such as Protein Data Bank, and the amino acidsequences common to each subgroup of FRs of VH and VL of humanantibodies (Sequences of Proteins of Immunological Interest, U.S. Dept.Health and Human Services, 1991).

As the CH for the antibody of the present invention, any CH ofantibodies can be used, so long as it belongs to human immunoglobulin(hereinafter referred to as hIg), but those of IgG class are preferred,and any one of subclasses further belonging to IgG such as γ1, γ2, γ3and γ4 can be used. Also, as CL of a human chimeric antibody, those of κclass or λ class can be used.

An example of the human CDR-grafted antibody of the present invention isa human CDR-grafted antibody or antibody fragment comprising CDR1, CDR2and CDR3 of VH of the antibody consisting of the amino acid sequencesrepresented by SEQ ID NOs: 5, 6 and 7, respectively; and/or CDR1, CDR2and CDR3 of VL of the antibody consisting of the amino acid sequencesrepresented by SEQ ID NOs:8, 9 and 10, respectively.

Preferred examples include a human CDR-grafted antibody, wherein the VHof the antibody comprises the amino acid sequence represented by SEQ IDNO: 13 or 14, and/or VL of the antibody comprises the amino acidsequence represented by SEQ ID NO: 15.

More Preferable examples include:

a human CDR-grafted antibody which comprises VH of the antibodycomprising an amino acid sequence in which at least one or more aminoacid residue selected from Ala at position 40, Gly at position 42, Lysat position 43, Gly at position 44, Lys at position 76 and Ala atposition 97 in the amino acid sequence represented by SEQ ID NO.13 isreplaced with another amino acid residue;

a human CDR-grafted antibody which comprises VH of the antibodycomprising an amino acid sequence in which at least one or more aminoacid residue selected from Thr at position 28 and Ala at position 97 inthe amino acid sequence represented by SEQ ID NO. 14 is replaced withanother amino acid residue;

a human CDR-grafted antibody which comprises VL of the antibodycomprising an amino acid sequence in which at least one or more aminoacid residue selected from Ile at position 2, Val at position 3, Gln atposition 50 and Val at position 88 in the amino acid sequencerepresented by SEQ ID NO. 15 is replaced with another amino acidresidue;

a human CDR-grafted antibody which comprises VH of the antibodycomprising an amino acid sequence in which at least one or more aminoacid residue selected from Ala at position 40, Gly at position 42, Lysat position 43, Gly at position 44, Lys at position 76, and Ala atposition 97 is replaced with another amino acid residue in the aminoacid sequence represented by SEQ ID NO.13, and VL of the antibodycomprising an amino acid sequence in which at least one or more aminoacid residue selected from Ile at position 2, Val at position 3, Gln atposition 50 and Val at position 88 in the amino acid sequencerepresented by SEQ ID NO. 15 is replaced with another amino acidresidue; and

A human CDR-grafted antibody which comprises VH of the antibodycomprising an amino acid sequence in which at least one or more aminoacid residue selected from Thr at position 28 and Ala at position 97 inthe amino acid sequence represented by SEQ ID NO. 14 is replaced withanother amino acid residue, and VL of the antibody comprising an aminoacid sequence in which at least one or more amino acid residue selectedfrom Ile at position 2, Val at position 3, Gln at position 50 and Val atposition 88 in the amino acid sequence represented by SEQ ID NO.15 isreplaced with another amino acid residue.

Still more preferable examples include a CDR-grafted antibody whichcomprises the heavy chain (H chain) variable region (V region) of theantibody comprising the amino acid sequence represented by SEQ ID NO:16or 17 and the light chain (L chain) V region of the antibody moleculecomprising the amino acid sequence represented by SEQ ID NO: 18, and thelike.

Also included within the scope of the present invention are antibodiesor antibody fragments which specifically react with CCR4 and consist ofamino acid sequences wherein one or more amino acid residues aredeleted, added, substituted or inserted in the above amino acidsequences.

The expression “one or more amino acid residues are deleted,substituted, inserted or added in the amino acid sequence in the presentinvention” means that one or more amino acids are deleted, substituted,inserted, or added at single or plural arbitrary position (s) in theamino acid sequence. Deletion, substitution, insertion and addition maybe caused in the same amino acid sequence simultaneously and amino acidresidues to be substituted, inserted or added may be either natural ornon-natural. Examples of the natural amino acid residues are L-alanine,L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine,L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine,L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan,L-tyrosine, L-valine and L-cysteine.

The followings are preferred examples of the amino acid residues capableof mutual substitution. The amino acid residues in the same group shownbelow can be mutually substituted.

-   Group A: leucine, isoleucine, norleucine, valine, norvaline,    alanine, 2-aminobutanoic acid, methionine, O-methylserine,    t-butylglycine, t-butylalanine, cyclohexylalanine-   Group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamic    acid, 2-aminoadipic acid, 2-aminosuberic acid-   Group C: asparagine, glutamine-   Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid,    2,3-diaminopropionic acid-   Group E: proline, 3-hydroxyproline, 4-hydroxyproline-   Group F: serine, threonine, homoserine-   Group G: phenylalanine, tyrosine

Specific examples of the human CDR-grafted antibody in the inventioninclude a human CDR-grafted antibody described in WO 03/18635, a humanCDR-grafted antibody prepared from monoclonal antibody 1G1, 2B10 or 10E4as described in WO 00/42074, a human CDR-grafted antibody prepared froma humanized antibody or monoclonal antibody 252Y or 252Z as described inWO 99/15666, a human CDR-grafted antibody prepared from a monoclonalantibody as described in U.S. Pat. No. 6,245,332.

The human antibody originally means an antibody naturally existing inthe human body. However, it also includes antibodies obtained from ahuman antibody phage library and human antibody-producing transgenicanimals prepared by the recent advance in genetic engineering, cellengineering and developmental engineering techniques, and the like.

With respect to the antibody naturally existing in the human body, forexample, human peripheral blood lymphocytes are isolated, infected withEB virus or the like for immortalization and cloning, wherebylymphocytes producing the antibody can be cultured, and the antibody canbe purified from the cultures.

The human antibody phage library is a library in which an antibody geneprepared from a human B cell is inserted into a phage gene to express anantibody fragment such as Fab or scFv on the surface of the phage. Alibrary in which mutation is artificially introduced can be used todevelop the library. From the library, a phage having a desired antigenbinding activity can be recovered using a binding activity to asubstrate having an antigen immobilized thereon as an index. Theantibody fragment can further be converted to a human antibody moleculecomprising two full length H chains and two full length L chains by aprotein engineering method.

The human antibody-producing transgenic animal means an animal in whicha human antibody gene is incorporated into cells. Specifically, a humanantibody-producing transgenic mouse prepared by introducing a humanantibody gene into a mouse ES cell, grafting the ES cell on an earlyembryo of the mouse and developing the same, and the like are mentioned.Regarding the method for preparing a human antibody from the humanantibody-producing transgenic animal, the human antibody can be producedand accumulated in a culture supernatant by culturing a humanantibody-producing hybridoma obtained by a hybridoma preparation methodgenerally carried out in cell fusion method.

Examples of transgenic non-human animals include cattle, sheep, goats,pigs, horses, mice, rats, chickens, monkeys, rabbits and the like.

An antibody fragment of the present invention includes Fab, Fab′,F(ab′)₂, scFv, Diabody, dsFv, and a peptide comprising CDR. An Fab is anantibody fragment having a molecular weight of about 50,000 and havingan antigen-binding activity, in which about a half of the N-terminalside of H chain and the full length L chain, among fragments obtained bytreating an IgG type antibody molecule with a protease, papain (cleavingat the amino acid residue at position 224 of the H chain), are boundtogether through a disulfide bond (S—S bond).

The Fab of the present invention can be obtained by treating the humanCDR-grafted antibody of the present invention which specifically reactswith human CCR4, with the protease, papain. Alternatively, the Fab maybe produced by inserting DNA encoding the Fab of the antibody into anexpression vector for prokaryote or eukaryote, and introducing thevector into a prokaryote or eukaryote to induce expression.

An F(ab′)₂ is an antibody fragment having a molecular weight of about100,000 and having an antigen-binding activity, which is slightly largerthan the Fab bound via a S—S bond at the hinge region, among fragmentsobtained by treating an IgG-type antibody molecule with a protease,pepsin (cleaving at the amino acid residue at position 234 of the Hchain).

The F(ab′)₂ of the present invention can be obtained by treating thehuman CDR-grafted antibody of the present invention which specificallyreacts with human CCR4 with the protease, pepsin. Alternatively, theF(ab′)₂ may be prepared by binding Fab′ fragments described below by athioether bond or a S—S bond.

An Fab′ is an antibody fragment with a molecular weight of approximately50,000 having antigen-binding activity, which is obtained by cleavingS—S bond at the hinge region of the above F(ab′)₂.

The Fab′ of the present invention can be obtained by treating theF(ab′)₂ of the present invention which specifically binds to human CCR4with a reducing agent, dithiothreitol. Alternatively, the Fab′ may beproduced by inserting DNA encoding the Fab′ of the human CDR-graftedantibody which specifically reacts with CCR4 into an expression vectorfor prokaryote or eukaryote, and introducing the vector into aprokaryote or eukaryote to induce expression.

An scFv is a VH-P-VL or VL-P-VH polypeptide in which one chain VH andone chain VL are linked by using an appropriate peptide linker (P) of 12or more amino acid residues and which has an antigen-binding activity.

The scFv of the present invention can be produced by obtaining cDNAsencoding the VH and VL of the human CDR-grafted antibody whichspecifically binds to CCR4, constructing DNA encoding the scFv,inserting the DNA into an expression vector for prokaryote or eukaryote,and introducing the expression vector into a prokaryote or eukaryote toinduce expression.

A diabody is an antibody fragment in which scFv having the same ordifferent antigen binding specificity forms a dimer, and has an divalentantigen binding activity to the same antigen or two specific antigenbinding activity to different antigens.

The diabody of the present invention, for example, a divalent diabodywhich specifically binds to CCR4, can be produced by obtaining cDNAsencoding VH and VL of the human CCR4-grafted antibody which bindsspecifically to CCR4, constructing DNA encoding scFv having apolypeptide linker of 3 to 10 amino acid residues, inserting the DNAinto an expression vector for prokaryote or an expression vector foreukaryote; and then introducing the expression vector into a prokaryoteor eukaryote to express the diabody.

A dsFV is an antibody fragment which is obtained by binding polypeptidesin which one amino acid residue of each of VH and VL is substituted witha cysteine residue and those cysteine residues are bound via a S—S bondbetween the cysteine residues. The amino acid residue which issubstituted with a cysteine residue can be selected based on athree-dimensional structure estimation of the antibody in accordancewith the method shown by Reiter et al. (Protein Engineering, 7, 697(1994)).

The dsFv of the present invention can be produced by obtaining cDNAsencoding VH and VL of the human CCR4-grafted antibody which specificallybinds to CCR4, constructing DNA encoding dsFv, inserting the DNA into anexpression vector for prokaryote or an expression vector for eukaryote,and then introducing the expression vector into a prokaryote oreukaryote to express the dsFv.

A peptide comprising CDR comprises one or more region of CDRs of VH andVL. The peptide comprising plural CDRs can be produced by bindingdirectly to or via an appropriate peptide linker.

The peptide comprising CDR of the present invention can be produced byconstructing cDNAs encoding CDR of VH and VL of the human CCR4-graftedantibody which specifically binds to CCR4, inserting the cDNAs into anexpression vector for prokaryote or an expression vector for eukaryote,and then by introducing the expression vector into a prokaryote oreukaryote to express the peptide. Also, the peptide comprising CDR canbe produced by a chemical synthesis method such as an Fmoc method(fluorenylmethoxycarbonyl method), a tBoc method (t-butyloxycarbonylmethod), or the like.

Agents used in the present invention include protein, agent having lowmolecular weight, and the like.

Examples of proteins include cytokines, antibodies and the like.

The cytokines include cytokines which activate the effector cells suchas NK cells, macrophages, monocytes, granulocytes, which areimmunocompetent cells. Specific examples of the cytokines includeinterleukin 2 (IL-2), IFN-α, IFN-γ, IL-12, IL-15, IL-18, IL-21,fractalkine, M-CSF, GM-CSF, G-CSF, TNF-α, TNF-β, IL-1α, IL-1β, and thelike.

Examples of antibodies include antibody, antibody fragment and fusionantibody which specifically react with surface markers of T cell.Specific antibodies include anti-CD3 antibody (Orthoclone), anti-CD4antibody, anti-CD5 antibody, anti-CD8 antibody, anti-CD30 antibody,anti-CD2 antibody, anti-CD25 antibody (Zenapax, Hoffmann-La Roche Inc.),anti-CD52 antibody (Campath, Ilex Oncology, Inc.), and the like.

Examples of the agent having low molecular weight of the presentinvention include amifostine (ethyol), cisplatin, dacarbazine (DTIC),dactinomycin, mechlorethamine (nitrogen mustard), streptozocin,cyclophosphamide, carmustine (BCNU), lomustine (CCNU), doxorubicin(adriamycin), doxorubicin lipo (doxil), gemcitabine (gemzar),daunorubicin, daunorubicin lipo (daunoxome), procarbazine, mitomycin,cytarabine, etoposide, Methotrexate, 5-fluorouracil, vinblastine,vincristine, bleomycin, paclitaxel (taxol), docetaxel (taxotere),aldesleukin, asparaginase, busulfan, carboplatin, cladribine,camptothecin, CPT-11, 10-hydroxy-7-ethyl-camptothecin (SN38),floxuridine, fludarabine, hydroxyurea, ifosfamide, idarubicin, mesna,irinotecan, mitoxantrone, topotecan, leuprolide, megestrol, melpharan,mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin,pipobroman, streptozocin, tamoxifen, teniposide, testolactone,thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil,prednisolone, vindesine, nimstine, semustin, capecitabine, tomudex,azacytidine, UFT, oxaloplatin, gefitinib (Iressa), imatinib (STI571),amsacrine, all-trans retinoic acid, thalidomide, bexarotene (targretin),dexamethasone, anastrozole (Alimidex), leuplin or combined use thereof.Preferable examples include vincristine, cyclophosphamide, etoposide,Methotrexate or combined use thereof.

When the above agent is administered in vivo solely at a high dose, fearof possible side effect may arose. However, in the present invention,the above agent can be used at a low dose by being combined with therecombinant antibody which specifically binds to CCR4 or the antibodyfragment thereof. Accordingly, in addition to the satisfactorytherapeutic effect, the side effect can be reduced.

The medicament of the invention can be used against cells in which CCR4is expressed. Tumor cells are preferable. Specifically, a hematopoieticorgan tumor is mentioned as the tumor.

The hematopoietic organ tumor includes acute leukemia, chronic leukemia,Hodgkin's disease, non-Hodgkin's disease and the like.

Examples of the acute leukemia include acute lymphatic leukemia and thelike, and examples of the acute lymphatic leukemia include pre-T cellacute lymphatic leukemia and the like.

The chronic leukemia includes chronic lymphatic leukemia. Examples ofthe chronic lymphatic leukemia include T cell chronic lymphaticleukemia, T cell pre-lymphatic leukemia, adult T cell leukemic lymphoma(ATL), Sezary syndrome and the like.

The non-Hodgkin's disease includes T/NK cell lymphoma. Examples of theT/NK cell lymphoma include pre-T lymphoblast lymphoma/leukemia, mature Tcell tumor and the like.

Examples of the mature T cell tumor include T cell pre-lymphocyteleukemia, T cell large granular lymphocyte leukemia, Sezary syndrome,mycosis fungoides, primary skin undifferentiated large cell lymphoma,subcutaneous phlegmon-like T cell lymphoma, intestinal disease-typebowel T cell lymphoma, liver/spleen yb T cell lymphoma,angioimmunoblastic T cell lymphoma, peripheral T cell lymphoma,undifferentiated large cell lymphoma, adult T cell leukemia/lymphoma andthe like.

The effect of the medicament of the present invention can be measured byan in vitro cytotoxic activity measuring method. As the in vitrocytotoxic activity measuring method, an ADCC measuring system ismentioned. ADCC can be measured by contacting target cells expressingCCR4 as an antigen with effector cells such as peripheral bloodmononuclear cells, monocytes, macrophages or granulocytes collected fromhumans or other animals, detecting a degree of damaged target cells, anddetermining the same. The degree of damaged target cells can be detectedby a ⁵¹Cr release method, a method for detecting an enzyme activity oftarget cells, a detecting method using a flow cytometer, or the like.The effect of the medicament of the present invention in the ADCCmeasuring system can be measured by adding the agent to the ADCCmeasuring system or exposing these agents to the target cells or theeffector cells or both of them for a prescribed period of time andobserving influences exerted on ADCC.

The effect of the medicament of the present invention may be examined bymeasuring an in vivo antitumor activity using animal models.

Examples of the animal models include xenograft models obtained bygrafting a culture cell line derived from a human cancer tissue inimmunodeficient mice such as nude mice, isograft models obtained bygrafting a cultured mouse cancer cell line in wild-type mice having anormal immune system, and the like.

The xenograft models can be prepared by grafting a human cancer cellline in various sites such as subcutaneous, intracutaneous,intraperitoneal and intravenous sites of immunodeficient mice such asnude mice.

The isograft models for evaluation of the medicament of the presentinvention can be prepared by introducing a CCR4 gene into a mouseculture cell line such as EL4 cell to form a CCR4-positive transformantand grafting this transformant into various sites of wild-type micehaving a normal immune system.

The effect of the medicament of the present invention can be evaluatedby comparing an effect of administration of the antibody alone or aneffect of administration of the agent alone with an effect of themedicament of the present invention using the animal models.

The medicament of the present invention can be administered alone, butit is generally preferred to provide it in the form of a pharmaceuticalpreparation produced by mixing it with one or more pharmaceuticallyacceptable carriers in accordance with any method well known in thetechnical field of pharmaceutics.

It is preferable to select a route of administration which is mosteffective in treatment. Examples include oral administration andparenteral administration, such as intraoral, tracheobronchial,intrarectal, subcutaneous, intramuscular and intravenous. In an proteinpreparation, intravenous administration is preferred.

Examples of the preparation suitable for the oral administration arespray, capsule, tablet, granule, syrup, emulsion, suppository,injection, ointment, tape and the like.

Liquid preparation such as emulsion and syrup can be produced usingwater, saccharides such as sucrose, sorbitol and fructose, glycols suchas polyethylene glycol and propylene glycol, oils such as sesame oil,olive oil and soybean oil, antiseptics such as p-hydroxybenzoate,flavors such as strawberry flavor and peppermint flavor and the like, asadditives.

Capsule, tablet, diluted powder, granule, and the like can be producedusing excipients such as lactose, glucose, sucrose and mannitol,disintegrating agents such as starch and sodium alginate, lubricantssuch as magnesium stearate and talc, binders such as polyvinyl alcohol,hydroxypropyl cellulose and gelatin, surfactants such as fatty acidester, plasticizers such as glycerol, as additives.

Examples of the preparation suitable for parenteral administration areinjection, suppository, air spray and the like.

Suppository is prepared using a carrier such as cacao butter,hydrogenated fat or carboxylic acid.

Air spray is prepared using the medicament as such or using, forexample, a carrier which does not stimulate the mouth and the airwaymucous membrane of a person to be administered, and which disperses themedicament into fine particles and makes the absorption easy.

Specific examples of the carrier are lactose and glycerol. Dependingupon the property of the medicament and the carrier used, it is possibleto prepare aerosol, dry powder, and the like. In addition, even in theparenteral preparation, components exemplified as additives in the oralpreparation may be added.

A dose or an administration schedule vary depending on a desiredtherapeutic effect, an administration method, a therapeutic period, anage, a body weight and the like. The dose of the antibody in oneadministration is usually from 0.1 to 20 mg/kg for an adult. The agentused in combination with the antibody is administered at a dose equal toor lower than the dose when the agent is used alone in clinic.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an enhancement effect of a cytokine against a cytotoxicactivity of an anti-CCR4 antibody. The ordinate shows a cytotocity. ▪indicates a cytotoxic activity without addition of cytokine, □ indicatesa cytotoxic activity in addition of IL-2, and a hatch indicates acytotoxic activity in addition of IL-15 respectively. A bar indicates astandard deviation.

FIG. 2 indicates an effect provided by combined use of an anti-CCR4antibody and vincristine against CCRF-CEM cells grafted in a nude mouse.The ordinate shows a V/V0 value. □ indicates a V/V0 value of a negativecontrol group, a slashed bar indicates a V/V0 value of KM 2760administration group, a gray indicates a V/V0 value of a vincristineadministration group, and ▪ indicates a V/V0 value of a group ofadministrating KM 2760 and vincristine in combination, respectively. Abar indicates a standard deviation.

FIG. 3 indicates an effect provided by combined use of an anti-CCR4antibody and cyclophosphamide against CCRF-CEM cells grafted in a nudemouse. The ordinate shows a V/V0 value. □ indicates a V/V0 value of anegative control group, a slashed bar indicates a V/V0 value of a KM2760 administration group, a gray indicates a V/V0 value of acyclophosphamide administration group, and ▪ indicates a V/V0 value of agroup of administration of KM 2760 and cyclophosphamide in combination,respectively. A bar indicates a standard deviation.

FIG. 4 indicates an effect provided by combined use of an anti-CCR4antibody and etopiside against CCRF-CEM cells grafted in a nude mouse.The ordinate shows a V/V0 value. □ indicates a V/V0 value of a negativecontrol group, a slashed bar indicates a V/V0 value of a KM 2760administration group, a gray indicates a V/V0 value of an etoposideadministration group, and ▪ indicates a V/V0 value of a groupadministrating KM 2760 and etoposide in combination, respectively. A barindicates a standard deviation.

FIG. 5 shows an effect provided by combined use of an anti-CCR4 antibodyand Methotrexate against CCRF-CEM cells grafted in a nude mouse. Theordinate shows a V/V0 value. □ indicates a V/V0 value of a negativecontrol group, a slashed bar indicates a V/V0 value of a KM 2760administration group, a gray indicates a V/V0 value of a Methotrexateadministration group, and ▪ indicates a V/V0 value of a groupadministrating of KM 2760 and Methotrexate in combination, respectively.A bar indicates a standard deviation.

FIG. 6 shows an effect provided by combined use of an anti-CCR4 antibodyand G-CSF against CCR4/EL4 cells grafted in C57BL/6 mouse. The abscissashows the number of days after grafting the tumor, the ordinate shows atumor volume respectively. X indicates a negative control group, ▴indicates a group of using KM 2760 alone, ● indicates a group of usingG-CSF alone, and □ indicates a group of combined use respectively. A barindicates a standard deviation.

FIG. 7 shows an effect provided by combined use of an anti-CCR4 antibodyand IFN-α against CCR4/EL4 cells grafted in C57BL/6 mouse. The ordinateshows a weight ratio of a liver. A bar indicates a standard deviation.

The present invention is described in detail below. The presentapplication claims the Convention priority from Japanese PatentApplication Nos. 2003-406590 and 2004-155141 filed Dec. 4, 2003 and May25, 2004, including the contents described in the specifications and/ordrawings of these applications.

BEST MODE FOR CARRYING OUT THE INVENTION Example 1

Effect of an Anti-CCR4 Antibody and a Cytokine in Combination in an InVitro Cytotoxic Activity

An effect of using anti-CCR4 human chimeric antibody KM 2760 (FERMBP-7054, WO 01/64754) and a cytokine in combination in an in vitrocytotoxic activity was measured by the following method.

(a) Preparation of an Effector Cell Suspension

A vein blood (50 mL) was collected from a healthy person, 0.5 mL ofheparin sodium (manufactured by Shimizu Seiyaku K.K.) was added, andthey were gently mixed. A mononuclear cell layer was separated fromusing a MONO-POLY separation solution (manufactured by DainipponPharmaceutical Co., Ltd.) according to the attached manual. After thelayer was subjected to centrifugation and washed with RPMI 1640-FCS (5)medium [RPMI 1640 medium containing 5% FCS (Gibco. BRL)] for threetimes, the cells were resuspended in the same medium to a concentrationof 3×10⁶ cells/ml to give an effector cell suspension.

(b) Stimulation of Effector Cells by a Cytokine

The effector cell suspension obtained in the above (a) was dispensed at50 μl/well in a 96-well U-bottom plate (manufactured by Falcon) in anamount of 50 μl/well. Further, either 50 μL of a 2 nm/mL IL-2(manufactured by Peprotech) solution diluted with RPMI 1640-FCS(5)medium, 50 μL of a 2 ng/mL of IL-15 (manufactured by Peprotech) solutionor 50 μL of RPMI 1640-FCS(5) as a negative control without addition of acytokine were added to separate samples, and were allowed to stand stillin a 5% CO₂ incubator for 3 days.

(c) Preparation of a Target Cell Suspension

CCR4/EL4 cells (WO01/64754) which are transformant tumor cells obtainedby introducing a human CCR4 gene into mouse thymoma cell line EL4 werecultured in RPMI 1640-FCS(10) medium [RPMI 1640 medium containing 10%FCS (manufactured by Gibco BRL)] containing 0.5 mg/mL of G418(manufactured by Nacalai Tesque) washed with RPMI 1640-FCS(5) mediumsubjected to centrifugation and suspension, and then adjusted to aconcentration of 2×10⁵ cells/mL with RPMI 1640-FCS(5) medium to form atarget cell suspension.

(d) Measurement of a Cytotoxic Activity

The target cell suspension (50 μL) prepared in the above (c) was addedto each well of a 96-well U-shaped bottom plate containing the effectorcells stimulated with the cytokine in the above (b) such that theconcentration became 1×10⁴ cells/well. At this time, the effectorcell:target cell ratio is 15:1. Further, KM 2760 was added such that thefinal concentration became 1 or 100 ng/mL to each well was allowed toreact, and at 37° C. for 4 hours. After the reaction, the plate wassubjected to centrifugation, and a lactic acid dehydrogenase(hereinafter referred to as LDH) activity in the supernatant wasmeasured by obtaining an absorbance data with CytoTox96 Non-RadioactiveCytotoxic activity Assay (manufactured by Promega) according to theattached manual. An absorbance data of target cell spontaneous releasewas obtained by the same procedure as above using RPMI 1640-FCS(5)medium in the same volume instead of the effector cell suspension andthe antibody solution, and absorbance data of effector cell spontaneousrelease was obtained by the same procedure as above using RPMI1640-FCS(5) medium in the same volume instead of the target cellsuspension and the antibody solution. With respect to an absorbance dataof target cell total release, a reaction was conducted using RPMI1640-FCS(5) medium in the same volume instead of the antibody solutionand the effector cell solution, 15 μL of a 9% Triton X-100 solution wasadded 45 minutes before completion of the reaction, and the sameprocedure as above was conducted to measure an LDH activity of thesupernatant. ADCC was obtained using the following formula.Cytotoxic activity (%)=[(absorbance of a specimen)−(absorbance ofeffector cell spontaneous release)−(absorbance of target cellspontaneous release)]/[(absorbance of target cell totalrelease)−(absorbance of target cell spontaneous release)]×100  (Formula1)

The results were shown in FIG. 1. The cytotoxic activity of KM 2760 wasincreased in a concentration-dependent manner. It was more increased byaddition of the cytokine. The results show that the cytotoxic activityof the anti-CCR4 antibody is enhanced by the cytokine that activates theeffector cells.

Example 2

Antitumor Effect Provided by Administrating an Anti-CCR4 Antibody andVincristine in Combination

CCRF-CEM cells (human T cell leukemia cell line) were suspended in RPMI1640 medium (Gibco BRL) at a concentration of 2×10⁸ cells/mL, and 100 μLof the suspension was grafted into the ventral skin of Balb/c nude mouse(Nippon Crea, male). On Day 15 after the cell grafting, a diameter of atumor was measured with calipers, and a tumor volume was calculatedusing the following formula.Tumor volume=short diameter×short diameter×long diameter×0.5  (Formula2)

Individuals having the tumor volume within the range of 140 to 342 mm³(on average 260 mm³) were selected, and grouped such that the average oftumor volume to be almost the same. Each of following agents A to D wasadministered to the mice. Incidentally, the grouping day was defined asDay 0.

A. Negative control group: No administration

B. Group of administering KM 2760 alone: 800 μg of KM2760 wasadministered per mouse on Day 0 and Day 4.

C. Group of administering vincristine (hereinafter referred to as VCR;Oncovin injection, Eli Lilly Japan K.K.) alone: 0.55 mg/kg of VCR wasadministered per mouse on Day 0.

D. Group of administering KM 2760 and VCR in combination: 0.55 mg/kg ofVCR was administered per mouse on Day 0, and 800 μg of KM 2760 wasadministered per mouse on Day 0 and Day 4.

The experiment was conducted with groups each consisting of five mice.Each of the agents was diluted with a physiological saline (OtsukaSeiyaku), and the diluent was administered from the tail vein. On Day10, the tumor volume was measured. The antitumor effect was evaluated bycomparing average values of a tumor volume change (V/V0) on Day 10 whenthe tumor volume on Day 0 in each group was defined as V0.

The average values of V/V0 in each group is shown in FIG. 2. As shown inFIG. 2, the administration of KM 2760 and VCR in combination exhibitedthe higher effect for suppressing growth than the administration of VCRor the antibody alone.

A value (T/C) obtained by dividing V/V0 of each group by V/V0 of thenegative control group is shown in Table 1. In comparison with atheoretical value of T/C when simply adding the pharmaceutical effectsof both KM 2760 and VCR, namely, a value obtained by multiplying T/Cs ofthe groups of administering the respective agents alone, actual T/C ofthe combined administration group (C in the table) exhibited the lowervalue (0.11) than 0.21, the theoretical value on Day 10. TABLE 1 T/C ofeach group A. Negative B. KM Theoretical control 2760 C. VCR D. KM2760 + VCR Value (B × C) 1 0.43 0.49 0.11 0.21

From the foregoing, it has been clarified that the administration of KM2760 and VCR in combination has the higher antitumor effect than theadministration of KM 2760 or VCR alone, and exhibits the synergisticeffect.

Example 3

Antitumor Effect Provided by Administrating an Anti-CCR4 Antibody andCyclophosphamide in Combination

CCRF-CEM cells (human T cell leukemia cell line) were suspended in RPMI1640 medium (Gibco BRL) at a concentration of 2×10⁸ cells/mL, and 100 μLof the suspension was grafted intradermally in the right flank of Balb/cnude mice (Nippon Crea, male). On Day 18 after the cell grafting, adiameter of a tumor was measured with calipers, and a tumor volume wascalculated using formula 2 in Example 2.

Individuals having the tumor volume within the range of 116 to 349 mm³(on average 219 mm³) were selected, and grouped such that the averagetumor volume to be almost the same. Each of the following agent A to Dwas administered to the mice. Incidentally, the grouping day was definedas Day 0.

A. Negative control group: No administration

B. Group of administering KM 2760 alone: 800 μg of KM2760 wasadministered per mouse on Day 0 and Day 4.

C. Group of administering cyclophosphamide (hereinafter referred to asCPA; Endoxan for injection, Baxter) alone: 65 mg/kg of CPA wasadministered per mouse on Day 0.

D. Group of administering KM 2760 and CPA in combination: 65 mg/kg ofCPA was administered per mouse on Day 0, and 800 μg of KM 2760 wasadministered per mouse on Day 0 and Day 4.

The experiment was conducted with groups each consisting of five mice.Each of the agents was diluted with a physiological saline (OtsukaSeiyaku), and the diluent was administered from the tail vein. On Day 4,the tumor volume was measured. The antitumor effect was evaluated bycomparing an average values of a tumor volume change (V/V0) on eachmeasurement day when the tumor volume on Day 0 in each group was definedas V0.

The chronological change in average values of V/V0 in each group isshown in FIG. 3. As shown in FIG. 3, the administration of KM 2760 andCPA in combination exhibited the higher effect for suppressing growththan the administration of CPA or the antibody alone.

A value (T/C) obtained by dividing V/V0 of each group by V/V0 of thenegative control group is shown in Table 2. In comparison with atheoretical value of T/C when simply adding the pharmaceutical effectsof both KM 2760 and CPA, namely, a value obtained by multiplying T/Cs ofthe groups of administering the respective agents alone, actual T/C ofthe combined administration group (D in the table) exhibited the lowervalue (0.35) than 0.39, the theoretical value. TABLE 2 T/C of each groupA. Negative B. KM Theoretical control 2760 C. CPA D. KM 2760 + CPA Value(B × C) 1.00 0.80 0.48 0.35 0.39

From the foregoing, it has been clarified that the administration ofKM2760 and CPA in combination has the higher antitumor effect than theadministration of KM2760 or CPA alone, and exhibits the synergisticeffect.

Example 4

Antitumor Effect Provided by Administrating an Anti-CCR4 Antibody andEtoposide in Combination

CCRF-CEM cells (human T cell leukemia cell line) were suspended in RPMI1640 medium (Gibco BRL) at a concentration of 2×10⁸ cells/mL, and 100 μLof the suspension was grafted into the ventral skin of Balb/c nude mice(Nippon Crea, male). On Day 17 after the cell grafting, a diameter of atumor was measured with calipers, and a tumor volume was calculatedusing formula 2 in Example 2.

Individuals having the tumor volume within the range of from 121 to 348mm³ (on average 195 mm³) were selected, and grouped such that theaverage tumor volume to be almost the same. Each of the following agentsA to D was then administered to the mice. Incidentally, the grouping daywas defined as Day 0.

A. Negative control group: No administration

B. Group of administering KM 2760 alone: 800 μg of KM2760 wasadministered per mouse on Day 0 and Day 4.

C. Group of administering etoposide (hereinafter referred to as VP-16;Lastet injection, Nippon Kayaku Co., Ltd.) alone: 10 mg/kg of VP-16 wasadministered per mouse for 5 days from Day 0 to Day 4.

D. Group of administering KM2760 and VP-16 in combination: 10 mg/kg ofVP-16 was administered per mouse for 5 days from Day 0 to Day 4, and 800μg of KM 2760 was administered per mouse on Day 0 and Day 4.

The experiment was conducted with groups each consisting of five mice.Each of the agents was diluted with a physiological saline solution(Otsuka Seiyaku), and the diluent was administered from the tail vein.On Day 7, the tumor volume was measured. The antitumor effect wasevaluated by comparing average values of a tumor volume change (V/V0) onDay 7 when the tumor volume on Day 0 in each group was defined as V0.

The average values of V/V0 in each group is shown in FIG. 4. As shown inFIG. 4, the administration of KM 2760 and VP-16 in combination exhibitedthe higher effect for suppressing growth than the administration ofVP-16 or the antibody alone.

A value (T/C) obtained by dividing V/V0 of each group by V/V0 of thenegative control group is shown in Table 3. In comparison with atheoretical value of T/C when simply adding the pharmaceutical effectsof both KM 2760 and VP-16, namely, a value obtained by multiplying T/Csof the groups of administering the respective agents alone, actual T/Cof the combined administration group (the value of D in the table)exhibited the lower value (0.38) than 0.46, the theoretical value. TABLE3 T/C of each group A. Negative D. KM Theoretical control B. KM 2760 C.VP-16 2760 + VP-16 Value (B × C) 1.00 0.65 0.71 0.38 0.46

From the foregoing, it has been clarified that the administration ofKM2760 and VP-16 in combination has the higher antitumor effect than theadministration of KM2760 or VP-16 alone, and exhibits the synergisticeffect.

Example 5

Antitumor Effect Provided by Administrating an Anti-CCR4 Antibody andMethotrexate in Combination

CCRF-CEM cells (human T cell leukemia cell line) were suspended in RPMI1640 medium (Gibco BRL) at a concentration of 2×10⁸ cells/mL, and 100 μLof the suspension was grafted into the ventral skin of Balb/c nude mice(CLEA Japan Inc. male). On Day 17 after the cell grafting, a diameter ofa tumor was measured with calipers, and a tumor volume was calculatedusing formula 2 in Example 2.

Individuals having the tumor volume within the range of 121 to 348 mm³(on average 195 mm³) were selected, and grouped such that the averagetumor volume to be almost the same. Each of the following agents A to Dwas administered to the mice. Incidentally, the grouping day was definedas Day 0.

A. Negative control group: No administration

B. Group of administering KM 2760 alone: 800 μg of KM2760 wasadministered per mouse on Day 0 and Day 4.

C. Group of administering Methotrexate (hereinafter referred to as MTX;Methotrexate injection solution, Nippon Weisledary K.K.) alone: 15 mg/kgof MTX was administered per mouse for 5 days from Day 0 to Day 4.

D. Group of administering KM 2760 and MTX in combination: 15 mg/kg ofMTX was administered per mouse for 5 days from Day 0 to Day 4, and 800μg of KM 2760 was administered per mouse on Day 0 and Day 4.

The experiment was conducted with groups each consisting of five mice.Each of the agents was diluted with a physiological saline solution(Otsuka Seiyaku), and the diluent was administered from the tail vein.On Day 7, the tumor volume was measured. The antitumor effect wasevaluated by comparing average values of a tumor volume change (V/V0)when the tumor volume on Day 0 in each group was defined as V0.

The chronological change in average values of V/V0 in each group isshown in FIG. 5. As shown in FIG. 5, the administration of KM 2760 andMTX in combination exhibited the higher effect for suppressing growththan the administration of MTX or the antibody alone.

A value (T/C) obtained by dividing V/V0 of each group by V/V0 of thenegative control group is shown in Table 4. In comparison with atheoretical value of T/C when simply adding the pharmaceutical effectsof both KM 2760 and MTX, namely, a value obtained by multiplying T/Cs ofthe groups of administering the respective agents alone, actual T/C ofthe combined administration group (the value of D in the table 4)exhibited the lower value (0.01) than 0.04, the theoretical value. TABLE4 T/C of each group A. Negative D. KM Theoretical control B. KM 2760 C.MTX 2760 + MTX Value (B × C) 1.00 0.65 0.06 0.01 0.04

From the foregoing, it has been clarified that the administration ofKM2760 and MTX in combination has the higher antitumor effect than theadministration of KM2760 or MTX alone, and exhibits the synergisticeffect.

Example 6

Antitumor Effect Provided by Administrating an Anti-CCR4 Human ChimericAntibody KM 2760 and G-CSF in Combination

CCR4/EL4 Cells (WO 01/64754) were suspended in RPMI 1640 medium (GibcoBRL) at a concentration of 1×10⁶ cells/mL, and 100 μL of the suspensionwas grafted into the right ventral skin of C57BL/6 mice (Nippon Crea,male, 8 weeks old). After, mice were grouped into A to D, each of theagents A to D were administered to each mouse. Incidentally, the day onwhich the tumor was grafted was defined as Day 0.

A. Negative control group: No administration

B. Group of administering KM 2760 alone: 100 μg of KM2760 wasintravenously administered per mouse on Day 0 and Day 4.

C. Group of administering G-CSF alone: 10 μg of G-CSF (Neuup Injection100, manufactured by Kyowa Hakko Kogyo Co., Ltd.) was subcutaneouslyadministered per mouse once a day for 10 days from 4 days before thetumor grafting (hereinafter referred to as Day-4) to Day 5. Theadministration site is near the hind limb on the right ventral portionwhich does not overlap with the tumor grafting site.

D. Group of administering KM2760 and G-CSF in combination: 100 μg of KM2760 was intravenously administered per mouse once a day on Day 0 andDay 4, and 10 μg of G-CSF was subcutaneously administered per mouse oncea day for 10 days from Day-4 to Day 5. The administration site is nearthe hind limb on the right ventral portion which does not overlap withthe tumor grafting site.

The experiment was conducted with group A consisting of 10 mice andgroups B, C and D each consisting of 7 mice. KM 2760 was diluted with acitrate buffer solution (10 mM citric acid, 150 mM sodium chloride, pH6), and G-CSF was diluted with a physiological saline (Otsuka Seiyaku)respectively. Each of the diluent was administered at 100 μL. A diameterof a tumor was measured with calipers chronologically from Day 0 of eachgroup. A tumor volume was calculated using formula 2 in Example 2.

Since the tumor death of the mouse was started in group A from Day 17on, the evaluation of the tumor volume was finished on Day 14.

The chronological change in averages value of the tumor volume in eachgroup is shown in FIG. 6. As shown in FIG. 6, antitumor effect was lowin groups B and C compared to untreated group A, whereas an outstandingantitumor effect was observed in group D.

T/C values on the final day of evaluation are shown in Table 5. Theantitumor effect (T/C value) in each group was evaluated by calculationusing the following formula 3 for comparison between the average valueof the tumor volume in group A and the average values of the tumorvolume in each group on the final evaluation day (Day 14).(T/C value)=(average value of tumor volume in each group on Day14)/average value of tumor volume in group A on Day 14).  (Formula 3)

In comparison with a theoretical value of T/C when simply adding thepharmaceutical effects of both KM 2760 and G-CSF, namely, a valueobtained by multiplying T/C values of the groups of administering therespective agents alone, actual T/C value of the combined administrationgroup (C in the table) exhibited the lower value (0.10) than 0.30, thetheoretical value. TABLE 5 Group A B C D Theoretical Value (B × C) T/Cvalue 1.0 0.48 0.63 0.10 0.30

From the foregoing, it has been clarified that the administration of KM2760 and G-CSF in combination has the higher antitumor effect than theadministration of KM 2760 or G-CSF alone, and exhibits the synergisticeffect.

Example 7

Antitumor Effect Provided by Administrating an Anti-CCR4 Human ChimericAntibody KM 2760 and IFN-α in Combination

CCR4/EL4 cells were suspended in RPMI 1640 medium (manufactured by GibcoBRL) at a concentration of 5× cells/mL, and 100 μl of the suspension wasgrafted into the tail vein of C57BL/6 mice (CLEA Japan Inc., male, 8weeks old). Further, mice were grouped into A to D, and each of theagents A to D were administered to the mice. Incidentally, the day onwhich the tumor was grafted was defined as Day 0.

A. Negative control group: No administration

B. Group of administering IFN-α alone: 5×10⁴ units of IFN-α (Universaltype I interferon, manufactured by PBL Biomedical Laboratories) wasintravenously administered per mouse once a day for 5 days from Day 1 toDay 5.

C. Group of administering KM 2760 alone: 0.5 μg of KM2760 wasintravenously administered per mouse once a day on Day 1 and Day 5.

D. Group of administering KM2760 and IFN-α in combination: 0.5 μg of KM2760 was intravenously administered per mouse once a day on Day 1 andDay 5, and 5×10⁴ units of IFN-α was intravenously administered per mouseonce a day for 5 days from Day 1 to Day 5.

The experiment was conducted with group A consisting of 7 mice andgroups B, C and D each consisting of 6 mice. KM 2760 was diluted with acitrate buffer solution (10 mM citric acid, 150 mM sodium chloride, pH6) and IFN-α was diluted PBS containing 0.1% bovine serum albuminrespectively. 100 μL of each of the diluents was administered. Theweight of all mice was measured on Day 14. After etherization andexsanguination, mice were brought to euthanasia by dislocation of thecervical spine. The liver was extracted, and the weight of the liver wasthen measured. A ratio of the liver weight to the weight of eachindividual (hereinafter referred to as a weight ration of a liver) wascalculated by percentage.

The antitumor effect was evaluated by comparing the liver weight ratio(average value of six mice) of untreated healthy mice measuredsimultaneously and the liver weight ratio of each group increased bymetastasis of tumor cells.

The liver weight ratio of each group is shown in FIG. 7. As shown inFIG. 7, antitumor effect was low in groups B and C compared to untreatedgroup A, whereas an outstanding antitumor effect was observed in groupD.

Further, the residual amount of tumor cells in the liver in each groupwas calculated as a T/C value using the following formula.T/C=(average values of a weight ration of a liver of each group−averagevalue of a weight ration of a liver of an untreated mouse) (averagevalue of a weight ration of a liver of a negative control group−averagevalue of a weight ration of a liver of an untreated mouse)  (Formula 4)

The resulting T/C values are shown in Table 6. In comparison with atheoretical value of T/C when simply adding the pharmaceutical effectsof both KM 2760 and IFN-α, namely, a value obtained by multiplying T/Cvalues of the groups of administering the respective agents alone,actual T/C value of the combined administration group (C in the table)exhibited the lower value (0.088) than 0.25, the theoretical value.TABLE 6 Group A B C D Theoretical Value (B × C) T/C value 1.0 0.48 0.520.088 0.25

From the foregoing, it has been clarified that the administration of KM2760 and IFN-α in combination has the higher antitumor effect than theadministration of KM 2760 or IFN-α alone, and exhibits the synergisticeffect.

Example 8

Antitumor Effect Provided by Administrating an Anti-CCR4 Human ChimericAntibody KM 2760 and M-CSF in Combination

CCR4/EL4 cells (WO 01/64754) were suspended in RPMI 1640 medium(manufactured by Gibco BRL) at a concentration of 1×10⁵ cells/mL, and200 μL of the suspension was grafted into the peritoneal cavity ofC57BL/6 mice (CLEA Japan Inc., male, 8 weeks old). After, mice wasgrouped into A to D, and each of the agents A to D was administered toeach mouse. The day on which the tumor was grafted was defined as Day 0.

A. Negative control group: No administration

B. Group of administering M-CSF alone: 100 μg of M-CSF (Leucoprol,manufactured by Kyowa Hakko Kogyo Co., Ltd.) was intraperitoneallyadministered per mouse twice a day from Day-3 to Day-1 and once a day onDay 0, seven times in total.

C. Group of administering KM 2760 alone: 50 μg of KM2760 wasintraperitoneally administered per mouse once a day on Day 0.

D. Group of administering KM 2760 and M-CSF in combination: 50 μg of KM2760 was intraperitoneally administered per mouse once a day on Day 0,and 100 μg was intraperitoneally administered per mouse twice a day fromDay-3 to Day-1 and once a day on Day 1, seven times in total.

The experiment was conducted with groups each consisting of 8 mice.Regarding the agents, KM 2760 was diluted with a citrate buffer (10 mMcitric acid, 150 mM sodium chloride, pH 6), and M-CSF was diluted with aphysiological saline solution (Otsuka Seiyaku) respectively. 100 μL ofeach of the diluents was administered. The antitumor effect wasevaluated by a ratio of average values of the number of survival days ofmice in each group to an average value of the number of survival days ofmice in the negative control group (hereinafter referred to as a lifeprolongation ratio). The number of survival days of each mouse and thelife prolongation ratio of each group are shown in Table 7. TABLE 7Number of Average number Life prolongation Group survival days ofsurvival days ratio A 17 18 18 19 19 20 20 22 19.3 1.00 B 17 17 18 18 1819 19 22 18.8 0.974 C 19 21 21 21 22 24 26 27 22.9 1.19 D 25 25 25 26 2626 27 >50 >28.8 >1.49 (theoretical value 1.16)

As shown in Table 7, In comparison with untreated Group A, no antitumoreffect was exhibited in group B, and antitumor effect was low in groupC, whereas an outstanding antitumor effect was observed in group D. Incomparison with a theoretical value of the life prolongation ratio whensimply adding the pharmaceutical effects of both KM 2760 and M-CSF,namely, a value obtained by multiplying the life prolongation ratios ofthe groups of administering the respective agents alone, the actual lifeprolongation ratio of the combined administration group (D in the table)exhibited the higher value (>1.49) than 1.16, the theoretical value.Incidentally, one mouse in group D was still alive even after anobservation period (Day 50).

From the foregoing, it has been clarified that the administration of KM2760 and M-CSF in combination has the higher antitumor effect than theadministration of KM 2760 or M-CSF alone, and exhibits the synergisticeffect.

INDUSTRIAL APPLICABILITY

A medicament comprising a combination of a recombinant antibody whichspecifically binds to human CC chemokine receptor 4 (CCR4) or anantibody fragment thereof and at least one agent is provided.

SEQ ID No. 13—Description of an artificial sequence: Antibody heavychain variable region amino acid sequence

SEQ ID No. 14—Description of an artificial sequence: Antibody heavychain variable region amino acid sequence

SEQ ID No. 15—Description of an artificial sequence: Antibody lightchain variable region amino acid sequence

SEQ ID No. 16—Description of an artificial sequence: Antibody heavychain variable region amino acid sequence

SEQ ID No. 17—Description of an artificial sequence: Antibody heavychain variable region amino acid sequence SEQ ID No. 18—Description ofan artificial sequence: Antibody light chain variable region amino acidsequence

1. A medicament comprising a combination of a recombinant antibody whichspecifically binds to human CC chemokine receptor 4 (CCR4) or anantibody fragment thereof and at least one pharmaceutically activeagents together with a pharmaceutically acceptable carrier. 2.(canceled)
 3. A kit, comprising a recombinant antibody whichspecifically binds to CCR4 or an antibody fragment thereof, and at leastone pharmaceutically active agent.
 4. The medicament according to claim1, wherein the pharmaceutically active agent is an antitumor drug.
 5. Aprocess of treating tumor in which CCR4 is expressed comprisingadministering the medicament according to any one of claims 4 or 7-20 toa patient in need thereof.
 6. The process according to the claim 5,wherein the tumor is a hematopoietic organ tumor.
 7. The medicamentaccording to claim 4, wherein the recombinant antibody whichspecifically binds to CCR4 or the antibody fragment thereof is anantibody which specifically binds to an extracellular region of CCR4 anddoes not show a reactivity to a human platelet.
 8. The medicamentaccording to the claim 7, wherein the recombinant antibody whichspecifically binds to the extracellular region of CCR4 or the antibodyfragment thereof does not have an activity of inhibiting binding of TARCor MDC as a CCR4 ligand to CCR4.
 9. The medicament according to claim 8,wherein the extracellular region is an extracellular region selectedfrom the group consisting of 1 to 39, 98 to 112, 176 to 206 and 271 to284 of an amino acid sequence represented by SEQ ID No.
 1. 10. Themedicament according to claim 8, wherein the extracellular region is anepitope existing at positions 2 to 29 of the amino acid sequencerepresented by SEQ ID No.
 1. 11. The medicament according to claim 8,wherein the extracellular region is an epitope existing at positions 13to 29 of the amino acid sequence represented by SEQ ID No.
 1. 12. Themedicament according to claim 8, wherein the extracellular region is anepitope existing at positions 13 to 25 of the amino acid sequencerepresented by SEQ ID No.
 1. 13. The medicament according to the claim12, wherein in the recombinant antibody which specifically binds to CCR4or the antibody fragment thereof, a binding activity to a peptidecomprising 13 to 25 of the amino acid sequence represented by SEQ ID No.1 in which at least one of tyrosine residues 16, 19, 20 and 22 issulfated is lower than a binding activity to a peptide comprising 13 to25 of the amino acid sequence represented by SEQ ID No.
 1. 14. Themedicament according to claim 13, wherein the recombinant antibody whichspecifically binds to the extracellular region of CCR4 or the antibodyfragment thereof is an antibody which specifically reacts with anepitope recognized by a monoclonal antibody which hybridoma KM 2160(FERM BP-10090) produces or an antibody fragment thereof.
 15. Themedicament according to claim 13, wherein the human recombinant antibodyis a human chimeric antibody or a human CDR-grafted antibody.
 16. Themedicament according to the claim 15, wherein the human chimericantibody comprises complementarity determining regions (CDRs) of a heavychain (H chain) variable region (V region) and a light chain (L chain) Vregion of a monoclonal antibody which specifically binds to CCR4. 17.The medicament according to claim 16, wherein the human chimericantibody comprises CDR1, CDR2 and CDR3 of a heavy chain (H chain)variable region (V region) of an antibody comprising amino acidsequences represented by SEQ ID Nos. 5, 6 and 7, respectively and/orCDR1, CDR2 and CDR3 of a light chain (L chain) variable region (Vregion) of an antibody comprising amino acid sequences represented bySEQ ID Nos. 8, 9 and 10, respectively.
 18. The medicament according toclaim 17, wherein the human chimeric antibody comprises a heavy chain (Hchain) variable region (V region) of an antibody molecule comprising anamino acid sequence represented by SEQ ID No. 11 and/or a light chain (Lchain) V region of an antibody molecule represented by SEQ ID No. 12.19. The medicament according to the claim 15, wherein the humanCDR-grafted antibody comprises complementarity determining regions(CDRs) of a heavy chain (H chain) variable region (V region) and a lightchain (L chain) V region of a monoclonal antibody which specificallybinds to CCR4.
 20. The medicament according to claim 19, wherein thehuman CDR-grafted antibody comprises CDR1, CDR2 and CDR3 of a heavychain (H chain) variable region (V region) of an antibody comprisingamino acid sequences represented by SEQ. ID Nos. 5, 6 and 7,respectively and/or CDR1, CDR2 and CDR3 of a light chain (L chain) Vregion comprising amino acid sequences represented by SEQ ID Nos. 8, 9and 10, respectively.
 21. The medicament according to any one of theclaims 15, 18 and 20, wherein the human CDR-grafted antibody comprises aheavy chain (H chain) variable region (V region) of an antibody moleculecomprising an amino acid sequence represented by SEQ ID No. 16 or 17and/or a light chain (L chain) V region of an antibody moleculerepresented by SEQ ID No.
 18. 22. The medicament according to claim 21,wherein the agent is a protein or an agent having low-molecular weight.23. The medicament according to the claim 22, wherein the protein is acytokine or an antibody.
 24. The medicament according to the claim 23,wherein the cytokine is a cytokine selected from G-CSF, M-CSF,interferon-α, IL-2 and IL-15.
 25. The medicament according to claim 24,wherein the agent having low-molecular weight is a chemotherapeuticagent or a hormone therapeutic agent.
 26. The medicament according tothe claim 25, wherein the chemotherapeutic agent is an agent selectedfrom vincristine, cyclophosphamide, etoposide and Methotrexate.